Hcv vaccines and methods for using the same

ABSTRACT

Improved anti-HCV immunogens and nucleic acid molecules that encode them are disclosed. Immunogens disclosed include those having consensus HCV genotype 1 a/1 b NS3 and NS4A. Pharmaceutical composition, recombinant vaccines comprising and live attenuated vaccines are disclosed as well methods of inducing an immune response in an individual against HCV are disclosed.

FIELD OF THE INVENTION

The present invention relates to improved HCV, improved methods forinducing immune responses, and for prophylactically and/ortherapeutically immunizing individuals against HCV.

BACKGROUND OF THE INVENTION

Hepatitis C (HCV) is a small enveloped, positive stranded RNA virus thatrepresents a major health burden worldwide with more than 170 millionindividuals currently infected [Thomson, B. J. and R. G. Finch,Hepatitis C virus infection. Clin Microbiol Infect, 2005. 11(2): p.86-94]. One of the most successful of all human viruses, HCVpreferentially infects heptocytes and is able to persist in the liversof up to 70% of all infected individuals [Bowen, D. O. and C. M. Walker,Adaptive immune responses in acute and chronic hepatitis C virusinfection. Nature, 2005. 436(7053): p. 946-52]. It is estimated that upto 30% of chronically infected individuals will develop progressiveliver disease, including cirrhosis and hepatocellular carcinoma (HCC)during their lifetime making HCV infection the leading causes of livertransplantation in the world. In addition, HCV and HBV infections areimplicated in 70% of all cases of HCC, which is the third leading causeof cancer deaths worldwide [Levrero, M., Viral hepatitis and livercancer: the case of hepatitis C. Oncogene, 2006. 25(27): p. 3834-47].

Due to the persistent nature of the virus, HCV infection can beextremely difficult and expensive to treat. Most infected individuals donot receive treatment. However, those that do, pay on average 17,700 to22,000 dollars for standard treatment protocols [Salomon, J. A., et al.,Cost-effectiveness of treatment for chronic hepatitis C infection in anevolving patient population. Jama, 2003. 290(2): p. 228-37]. Genotype 1infection, the most prevalent in Europe and North America, has thepoorest prognosis with as little as 42% of individuals responding tostandard treatments [Manns, M. P., et al., Peginterferon alfa-2b plusribavirin compared with interferon alfa-2b plus ribavirin for initialtreatment of chronic hepatitis C: a randomised trial. Lancet, 2001.358(9286): p. 958-65].

Therefore, the high prevalence of infection, lack of effectivetreatments and economic burden of chronic HCV, illustrates the urgentneed for the development of novel immune therapy strategies to combatthis disease. Currently there is no prophylactic or therapeutic vaccinefor HCV, however there is evidence that natural and protective immunityto HCV exists [Weiner, A. J., et al., Intrahepatic genetic inoculationof hepatitis C virus RNA confers cross-protective immunity. J Virol,2001. 75(15): p. 7142-8; Bassett, S. E., et al., Protective immuneresponse to hepatitis C virus in chimpanzees rechallenged followingclearance of primary infection. Hepatology, 2001. 33(6): p. 1479-87;Lanford, R. E., et al., Cross-genotype immunity to hepatitis C virus. JVirol, 2004. 78(3): p. 1575-81]. In the majority of cases, convalescenthumans are not protected against acute HCV infection, but rather, theyare protected from the progression of infection to a chronic state[Houghton, M. and S. Abrignani, Prospects for a vaccine against thehepatitis C virus. Nature, 2005. 436(7053): p. 961-6]. Since it is thechronic state of infection that is mainly associated with HCVpathogenicity, this argues for the feasibility of a vaccine approach tocontrol or treat this infection.

Understanding the adaptive immunity to this virus is critical fordesigning strategies, such as DNA vaccines, to combat viral infection.Although virus-specific antibodies are detected within 7-8 weeks postHCV infection [Pawlotsky, J. M., Diagnostic tests for hepatitis C. JHepatol, 1999. 31 Suppl 1: p. 71-9] they do not protect againstreinfection [Farci, P., et al., Lack of protective immunity againstreinfection with hepatitis C virus. Science, 1992. 258(5079): p. 135-40;Lai, M. E., et al., Hepatitis C virus in multiple episodes of acutehepatitis in polytransfused thalassaemic children. Lancet, 1994.343(8894): p. 388-90] and can be completely absent following theresolution of infection [Cooper, S., et al., Analysis of a successfulimmune response against hepatitis C virus. Immunity, 1999. 10(4): p.439-49; Post, J. J., et al., Clearance of hepatitis C viremia associatedwith cellular immunity in the absence of seroconversion in the hepatitisC incidence and transmission in prisons study cohort. Infect Dis, 2004.189(10): p. 1846-55]. Instead, infected individuals that mount an early,multi-specific, intrahepatic CD4+ helper and CD8+ cytotoxic T-cellresponse can eliminate HCV infection [Lechner, F., et al., Analysis ofsuccessful immune responses in persons infected with hepatitis C virus.J Exp Med, 2000. 191(9): p. 1499-512; Gerlach, J. T., et al., Recurrenceof hepatitis C virus after loss of virus-specific CD4(+) T-cell responsein acute hepatitis C. Gastroenterology, 1999. 117(4): p. 933-41; Thimme,R., et al., Determinants of viral clearance and persistence during acutehepatitis C virus infection. J Exp Med, 2001. 194(10): p. 1395-406;Grakoui, A., et al., HCV persistence and immune evasion in the absenceof memory T cell help. Science, 2003. 302(5645): p. 659-62]. In fact, ithas been shown that an important correlate to resolution of acuteinfection is a strong T cell response against the structural proteins ofthe virus, in particular the NS3 protein [Missale, G., et al., Differentclinical behaviors of acute hepatitis C virus infection are associatedwith different vigor of the anti-viral cell-mediated immune response. JClin Invest, 1996. 98(3): p. 706-14; Diepolder, H. M., et al., Possiblemechanism involving T-lymphocyte response to non-structural protein 3 inviral clearance in acute hepatitis C virus infection. Lancet, 1995.346(8981): p. 1006-7]. The correlation of NS3-specific T cell responsesto resolution of acute infection, in addition to its low geneticvariably and relative large size makes the NS3 protein of HCV anattractive target for T-cell based DNA vaccines.

DNA vaccines have many conceptual advantages over more traditionalvaccination methods, such as live attenuated viruses and recombinantprotein-based vaccines. DNA vaccines are safe, stable, easily produced,and well tolerated in humans with preclinical trials indicating littleevidence of plasmid integration [Martin, T., et al., Plasmid DNA malariavaccine: the potential for genomic integration after intramuscularinjection. Hum Gene Ther, 1.999. 10(5): p. 759-68; Nichols, W. W., etal., Potential DNA vaccine integration into host cell genome. Ann N YAcad Sci, 1995. 772: p. 30-9]. In addition, DNA vaccines are well suitedfor repeated administration due to the fact that efficacy of the vaccineis not influenced by pre-existing antibody titers to the vector[Chattergoon, M., Boyer, and D. B. Weiner, Genetic immunization: a newera in vaccines and immune therapeutics. FASEB J, 1997. 11(10): p.753-63]. However, one major obstacle for the clinical adoption of DNAvaccines has been a decrease in the platforms immunogenicity when movingto larger animals [Liu, M. A. and J. B. Ulmer, Human clinical trials ofplasmid DNA vaccines. Adv Genet, 2005. 55: p. 25-40]. Recenttechnological advances in the engineering of DNA vaccine immunogen, suchhas codon optimization, RNA optimization and the addition ofimmunoglobulin leader sequences have improved expression andimmunogenicity of DNA vaccines [Andre, S., et al., Increased immuneresponse elicited by DNA vaccination with a synthetic gp120 sequencewith optimized codon usage. J Virol, 1998. 72(2): p. 1497-503; Deml, L.,et al., Multiple effects of codon usage optimization on expression andimmunogenicity of DNA candidate vaccines encoding the humanimmunodeficiency virus type 1 Gag protein. J Virol, 2001. 75(22): p.10991-1001; Laddy, D. J., et al., Immunogenicity of novelconsensus-based DNA vaccines against avian influenza. Vaccine, 2007.25(16): p. 2984-9; Frelin, L., et al., Codon optimization and mRNAamplification effectively enhances the immunogenicity of the hepatitis Cvirus nonstructural 3/4A gene. Gene Ther, 2004. 11(6): p. 522-33], aswell as, recently developed technology in plasmid delivery systems suchas electroporation [Hirao, L. A., et al., Intradermal/subcutaneousimmunization by electroporation improves plasmid vaccine delivery andpotency in pigs and rhesus macaques. Vaccine, 2008. 26(3): p. 440-8;Luckay, A., et al., Effect of plasmid DNA vaccine design and in vivoelectroporation on the resulting vaccine-specific immune responses inrhesus macaques. J Virol, 2007. 81(10): p. 5257-69; Ahlen, G., et al.,In vivo electroporation enhances the immunogenicity of hepatitis C virusnonstructural 3/4A DNA by increased local DNA uptake, proteinexpression, inflammation, and infiltration of CD3+ T cells. J Immunol,2007. 179(7): p. 4741-53]. In addition, studies have suggested that theuse of consensus immunogens may be able to increase the breadth of thecellular immune response as compared to native antigens alone [Yan., J.,et al., Enhanced cellular immune responses elicited by an engineeredHIV-1 subtype 13 consensus-based envelope DNA vaccine. Mol Ther, 2007.15(2): p. 411-21; Rolland, M., et al., Reconstruction and function ofancestral center-of-tree human immunodeficiency virus type 1 proteins. JVirol, 2007. 81(16): p. 8507-14].

DNA vaccines encoding HCV NS3 and NS4 are disclosed in an onlinepublication on the website www.elsevier.com/locate/vaccine in an articleby Lang. K. A. et al, Vaccine XX (2008) XXX-XXX.

There remains a need for an effective vaccine against CV. There remainsa need for effective methods of treating individuals infected with HCV.

SUMMARY OF THE INVENTION

Proteins comprising consensus HCV genotype 1a/1b NS3 and NS4A amino acidsequences and nucleic acid molecules that comprising a nucleotidesequence encoding such proteins are provided. These nucleic acidconstructs and proteins encoded thereby provide improved immunogenictargets against which an anti-HCV immune response can be generated.

Constructs which encode such proteins sequences, vaccines which comprisesuch proteins and/or nucleic acid molecules that encode such proteins,and methods of inducing anti-HCV immune responses are also provided.

Nucleic acid molecules comprising a nucleotide sequence selected fromthe group consisting of: SEQ ID NO:1; fragments of SEQ ID NO:1;sequences having at least 90% homology to SEQ ID NO:1; and fragments ofsequences having at least 90% homology to SEQ ID NO:1.

The present invention relates to nucleic acid molecules comprising anucleotide sequence selected from the group consisting of: nucleotidesequences that encode SEQ ID NO:2; nucleotide sequences that encode anamino acid sequences having at least 90% homology to SEQ ID NO:2;fragments of nucleotide sequences that encode SEQ ID NO:2; fragments ofa nucleotide sequence that encode an amino acid sequence having at least90% homology to SEQ ID NO:2.

The present invention further provides pharmaceutical compositionscomprising such nucleic acid molecules and their use in methods ofinducing an immune response in an individual against HCV that compriseadministering to an individual a composition comprising such nucleicacid molecules.

The present invention further provides recombinant vaccine comprisingsuch nucleic acid molecules and their use in methods of inducing animmune response in an individual against HCV that comprise administeringto an individual such a recombinant vaccine.

The present invention further provides live attenuated pathogenscomprising such nucleic acid molecules and their use in methods ofinducing an immune response in an individual against HCV that compriseadministering to an individual such live attenuated pathogens.

Live Attenuated Pathogen

The present invention further provides proteins comprising amino acidsequences selected from the group consisting of: SEQ ID NO:2, sequenceshaving at least 90% homology to SEQ ID NO:2; fragments of SEQ ID NO:2;and fragments of sequences having at least 90% homology to SEQ ID NO:2.

The present invention further provides pharmaceutical compositionscomprising such proteins and their use in methods of inducing an immuneresponse in an individual against HCV that comprise administering to anindividual a composition comprising such proteins.

The present invention further provides recombinant vaccines comprisingsuch proteins and their use in methods of inducing an immune response inan individual against HCV that comprise administering to an individualsuch a recombinant vaccine.

The present invention further provides live attenuated pathogenscomprising such proteins and their use in methods of inducing an immuneresponse in an individual against HCV that comprise administering to anindividual such live attenuated pathogens.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Phylogenetic analysis of pConNS3/NS4A's genotype 1a/1b consensussequence of NS3 as compared to individual genotype 1a and genotype 1bsequences for NS3. The genotype 1a/1b consensus sequence for NS3 wasobtained from fifteen different HCV genotype 1a sequences and twenty-sixdifferent HCV genotype 1b sequences. The star represents the NS3consensus sequence relative to its forty-one different componentsequences.

FIG. 2: Plasmid map and sequence of pConNS3/NS4A. The sequences for theIgE leader, endoproteolytic cleavage site and C-terminal HA tag areunderlined.

FIG. 3: Detection of pConNS3/NS4A expression via immunofluorescence(400×). Huh7.0 were transiently transfected with pConNS3/NS4A andexpression of the gene product was detected using a monoclonal antibodyagainst the C-terminal HA tag.

FIG. 4: pConNS3/NS4A induces strong NS3- and NS4-specific T cellresponses in C57BL/6 mice. The number of NS3- and NS4-specific IFN-gammaspot forming units (SFU) per million splenocytes was determined throughIFN-gamma ELISpot assays. (A) Five groups of mice, three mice per group,were immunized intramuscularly with either pVAX (negative control) orfour different doses of pConNS3/NS4A: 5 ug, 12.5 ug, 25 ug or 50 ugfollowed by electroporation. Splenocytes were isolated from each mouse,pooled according to group and stimulated with five different pools ofoverlapping peptides spanning the entire length of the pConNS3/NS4Aprotein sequence. (B) Matrix epitope mapping of pConNS3/NS4A.Splenocytes were isolated from C57BL/6 mice immunized with 12.5 ug ofpConNS3/NS4A and stimulated with 21 different pools of overlappingpConNS3/NS4A peptides with each peptide represented in two of the 21pools. The dominant epitope was identified using IFN-gamma ELISpotassays as described above.

FIG. 5: pConNS3/NS4A induces strong NS3- and NS4-specific T cellresponses in Rhesus Macaques. (A) Immunization schedule. Five RhesusMacaques were immunized intramuscularly with 1 mg pConNS3/NS4A followingby electroporation. The monkeys received two immunizations, four weeksapart. (B) Responses were measured once before the first immunizationand two weeks following each immunization. The number of NS3- andNS4-specific IFN-gamma spot forming units (SFU) per million PBMCs wasdetermined through IFN-gamma ELISpot assays.

FIG. 6: pConNS3/NS4A induces broad cellular immune responses in RhesusMacaques. Shown are the individual responses of the five monkeys to eachof the five peptide pools, before immunization and two weeks followingeach immunization. The number of NS3- and NS4-specific IFN-gamma spotforming units (SFU) per million PBMCs was determined through IFN-gammaELISpot assays.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein, the phrase “stringent hybridization conditions” or“stringent conditions” refers to conditions under which a nucleic acidmolecule will hybridize another a nucleic acid molecule, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. Since thetarget sequences are generally present in excess, at Tm, 50% of theprobes are occupied at equilibrium. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes,primers or oligonucleotides (e.g. 10 to 50 nucleotides) and at leastabout 60° C. for longer probes, primers or oligonucleotides. Stringentconditions may also be achieved with the addition of destabilizingagents, such as formamide.

Sequence homology for nucleotides and amino acids may be determinedusing FASTA, BLAST and Gapped BLAST (Altschul et al., Nuc. Acids Res.,1997, 25, 3389, which is incorporated herein by reference in itsentirety) and PAUP*4.0b10 software (D, L. Swofford, Sinauer Associates,Massachusetts). “Percentage of similarity” is calculated usingPAUP*4.0b10 software (D. L. Swofford, Sinauer Associates,Massachusetts). The average similarity of the consensus sequence iscalculated compared to all sequences in the phylogenic tree.

Briefly, the BLAST algorithm, which stands for Basic Local AlignmentSearch Tool is suitable for determining sequence similarity (Altschul etal., J. Mol. Biol., 1990, 215, 403-410, which is incorporated herein byreference in its entirety). Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/). This algorithm involvesfirst identifying high scoring sequence pair (HSPs) by identifying shortwords of length W in the query sequence that either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to find HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Extension for the word hits in each direction are haltedwhen: 1) the cumulative alignment score falls off by the quantity X fromits maximum achieved value; 2) the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or 3) the end of either sequence is reached. The Blastalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The Blast program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad.Sci. USA, 1992, 89, 10915-10919, which is incorporated herein byreference in its entirety) alignments (B) of 50, expectation (E) of 10,M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlinet al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, which isincorporated herein by reference in its entirety) and Gapped BLASTperform a statistical analysis of the similarity between two sequences.One measure of similarity provided by the BLAST algorithm is thesmallest sum probability (P(N)), which provides an indication of theprobability by which a match between two nucleotide sequences wouldoccur by chance. For example, a nucleic acid is considered similar toanother if the smallest sum probability in comparison of the testnucleic acid to the other nucleic acid is less than about 1, preferablyless than about 0.1, more preferably less than about 0.01, and mostpreferably less than about 0.001.

As used herein, the term “genetic construct” refers to the DNA or RNAmolecules that comprise a nucleotide sequence which encodes protein. Thecoding sequence includes initiation and termination signals operablylinked to regulatory elements including a promoter and polyadenylationsignal capable of directing expression in the cells of the individual towhom the nucleic acid molecule is administered.

As used herein, the term “expressible form” refers to gene constructsthat contain the necessary regulatory elements operable linked to acoding sequence that encodes a protein such that when present in thecell of the individual, the coding sequence will be expressed.

Improved vaccine are disclosed which arise from a multi-phase strategyto enhance cellular immune responses induced by immunogens. Modifiedconsensus sequences were generated. Genetic modifications includingcodon optimization, RNA optimization, and the addition of a highefficient immunoglobin leader sequence are also disclosed. The novelconstruct has been designed to elicit stronger and broader cellularimmune responses than a corresponding codon optimized immunogens.

The improved HCV vaccines are based upon proteins and genetic constructsthat encode proteins with epitopes that make them particularly effectiveas immunogens against which anti-HCV can be induced. Accordingly,vaccines may induce a therapeutic or prophylactic immune response. Insome embodiments, the means to deliver the immunogen is a DNA vaccine, arecombinant vaccine, a protein subunit vaccine, a composition comprisingthe immunogen, an attenuated vaccine or a killed vaccine. In someembodiments, the vaccine comprises a combination selected from thegroups consisting of: one or more DNA vaccines, one or more recombinantvaccines, one or more protein subunit vaccines, one or more compositionscomprising the immunogen, one or more attenuated vaccines and one ormore killed vaccines.

According to some embodiments, a vaccine is delivered to an individualto modulate the activity of the individual's immune system and therebyenhance the immune response against HCV. When a nucleic acid moleculesthat encodes the protein is taken up by cells of the individual thenucleotide sequence is expressed in the cells and the protein arethereby delivered to the individual. Methods of delivering the codingsequences of the protein on nucleic acid molecule such as plasmid, aspart of recombinant vaccines and as part of attenuated vaccines, asisolated proteins or proteins part of a vector are provided.

Compositions and methods are provided which prophylactically and/ortherapeutically immunize an individual against HCV.

Compositions for delivering nucleic acid molecules that comprise anucleotide sequence that encodes the immunogen are operably linked toregulatory elements. Compositions may include a plasmid that encodes theimmunogen, a recombinant vaccine comprising a nucleotide sequence thatencodes the immunogen, a live attenuated pathogen that encodes a proteinof the invention and/or includes a protein of the invention; a killedpathogen includes a protein of the invention; or a composition such as aliposome or subunit vaccine that comprises a protein of the invention.The present invention further relates to injectable pharmaceuticalcompositions that comprise compositions.

SEQ ID NO:1 comprises a nucleotide sequence that encodes an HCV genotype1a/1b consensus immunogen of HCV proteins NS3/NS4A. SEQ ID NO:1 furthercomprises an IgE leader sequence linked to the nucleotide sequence thatencodes an HCV genotype 1a/1b consensus immunogen of HCV proteinsNS3/NS4A. SEQ ID NO:2 comprises the amino acid sequence for the HCVgenotype 1a/1b consensus immunogen of HCV proteins NS3/NS4A. SEQ ID NO:2further comprises an IgE leader sequence linked to a consensus immunogensequence. The IgE leader sequence is SEQ ID NO:4 and may be encoded bySEQ ID NO:3.

In some embodiments, vaccines of include SEQ ID NO:4, or a nucleic acidmolecule that encodes SEQ ID NO:4. In some embodiments, vaccinespreferably comprise SEQ ID NO:2 or a nucleic acid molecule that encodesit. In some embodiments, vaccines preferably comprise SEQ ID NO:1.Vaccines preferably include the IgE leader sequence SEQ ID NO:4 ornucleic acid sequence which encodes the same.

Fragments of SEQ ID NO:1 may comprise 90 or more nucleotides. In someembodiments, fragments of SEQ ID NO:1 may comprise 180 or morenucleotides; in some embodiments, 270 or more nucleotides; in someembodiments 360 or more nucleotides; in some embodiments, 450 or morenucleotides; in some embodiments 540 or more nucleotides; in someembodiments, 630 or more nucleotides; in some embodiments, 720 or morenucleotides; in some embodiments, 810 or more nucleotides; in someembodiments, 900 or more nucleotides; in some embodiments, 990 or morenucleotides; in some embodiments, 1080 or more nucleotides; in someembodiments, 1170 or more nucleotides; in some embodiments, 1260 or morenucleotides; in some embodiments, 1350 or more nucleotides in someembodiments, 1440 or more nucleotides; in some embodiments, 1530 or morenucleotides; in some embodiments, 1620 or more nucleotides; in someembodiments, 1710 or more nucleotides; in some embodiments, 1800 or morenucleotides; in some embodiments, 1890 or more nucleotides; in someembodiments, 1980 or more nucleotides; and in some embodiments, 2070 ormore nucleotides. In some embodiments, fragments of SEQ ID NO:1 maycomprise coding sequences for the IgE leader sequences. In someembodiments, fragments of SEQ ID NO:1 do not comprise coding sequencesfor the IgE leader sequences. Fragments may comprise fewer than 180nucleotides, in some embodiments fewer than 270 nucleotides, in someembodiments fewer than 360 nucleotides, in some embodiments fewer than450 nucleotides, in some embodiments fewer than 540 nucleotides, in someembodiments fewer than 630 nucleotides, and in some embodiments fewerthan 675 nucleotides.

Fragments of SEQ ID NO:2 may comprise 30 or more amino acids. In someembodiments, fragments of SEQ ID NO:2 may comprise 60 or more aminoacids; in some embodiments, 90 or more amino acids; in some embodiments,120 or more amino acids; in some embodiments; 150 or more amino acids;in some embodiments 180 or more amino acids; in some embodiments, 210 ormore amino acids; in some embodiments, 240 or more amino acids; in someembodiments, 270 or more amino acids; in some embodiments, 300 or moreamino acids; in some embodiments, 330 or more amino acids; in someembodiments, 360 or more amino acids; in some embodiments, 390 or moreamino acids; in some embodiments, 420 or more amino acids; in someembodiments, 450 or more amino acids; in some embodiments, 480 or moreamino acids; in some embodiments, 510 or more amino acids; in someembodiments, 540 or more amino acids; in some embodiments, 570 or moreamino acids; in some embodiments, 600 or more amino acids; in someembodiments, 630 or more amino acids; in some embodiments, 660 or moreamino acid; and in some embodiments, 690 or more amino acids. Fragmentsmay comprise fewer than 90 amino acids, in some embodiments fewer than120 amino acids, in some embodiments fewer than 150 amino acids, in someembodiments fewer than 180 amino acids, in some embodiments fewer than210 amino acids, in some embodiments fewer than 240 amino acids, in someembodiments fewer than 270 amino acids, in some embodiments fewer than300 amino acids, in some embodiments fewer than 330 amino acids, in someembodiments fewer than 360 amino acids, in some embodiments fewer than390 amino acids, in some embodiments fewer than 420 amino acids, in someembodiments fewer than 450 amino acids, in some embodiments fewer than480 amino acids, in some embodiments fewer than 540 amino acids, in someembodiments fewer than 600 amino acids, in some embodiments fewer than660 amino acids, and in some embodiments fewer than 690 amino acids.

SEQ ID NO:5 comprises a nucleotide sequence that encodes an HCV genotype1a/1b consensus immunogen of HCV proteins NS3/NS4A. SEQ ID NO:6comprises the amino acid sequence for the HCV genotype 1a/1b consensusimmunogen of HCV proteins NS3/NS4A.

Fragments of SEQ ID NO:5 may comprise 90 or more nucleotides. In someembodiments, fragments of SEQ ID NO:5 may comprise 180 or morenucleotides; in some embodiments, 270 or more nucleotides; in someembodiments 360 or more nucleotides; in some embodiments, 450 or morenucleotides; in some embodiments 540 or more nucleotides; in someembodiments, 630 or more nucleotides; in some embodiments, 720 or morenucleotides; in some embodiments, 810 or more nucleotides; in someembodiments, 900 or more nucleotides; in some embodiments, 990 or morenucleotides; in some embodiments, 1080 or more nucleotides; in someembodiments, 1170 or more nucleotides; in some embodiments, 1260 or morenucleotides; in some embodiments, 1350 or more nucleotides in someembodiments, 1440 or more nucleotides; in some embodiments, 1530 or morenucleotides; in some embodiments, 1620 or more nucleotides; in someembodiments, 1710 or more nucleotides; in some embodiments, 1800 or morenucleotides; in some embodiments, or more nucleotides. Fragments maycomprise fewer than 180 nucleotides, in some embodiments fewer than 270nucleotides, in some embodiments fewer than 360 nucleotides, in someembodiments fewer than 450 nucleotides, in some embodiments fewer than540 nucleotides, in some embodiments fewer than 630 nucleotides, and insome embodiments fewer than 675 nucleotides.

Fragments of SEQ ID NO:6 may comprise 30 or more amino acids. In someembodiments, fragments of SEQ ID NO:6 may comprise 60 or more aminoacids; in some embodiments, 90 or more amino acids; in some embodiments,120 or more amino acids; in some embodiments; 150 or more amino acids;in some embodiments 180 or more amino acids; in some embodiments, 210 ormore amino acids; in some embodiments, 240 or more amino acids; in someembodiments, 270 or more amino acids; in some embodiments, 300 or moreamino acids; in some embodiments, 330 or more amino acids; in someembodiments, 360 or more amino acids; in some embodiments, 390 or moreamino acids; in some embodiments, 420 or more amino acids; in someembodiments, 450 or more amino acids; in some embodiments, 480 or moreamino acids. Fragments may comprise fewer than 90 amino acids, in someembodiments fewer than 120 amino acids, in some embodiments fewer than150 amino acids, in some embodiments fewer than 180 amino acids, in someembodiments fewer than 210 amino acids, in some embodiments fewer than240 amino acids, in some embodiments fewer than 270 amino acids, in someembodiments fewer than 300 amino acids, in some embodiments fewer than330 amino acids, in some embodiments fewer than 360 amino acids, in someembodiments fewer than 390 amino acids, in some embodiments fewer than420 amino acids, and in some embodiments fewer than 450 amino acids.

According to some embodiments, methods of inducing an immune response inindividuals against an immunogen comprise administering to theindividual the amino acid sequence for the HCV genotype 1a/1b consensusimmunogen of HCV proteins NS3/NS4A, or functional fragments thereof, orexpressible coding sequences thereof. Some embodiments comprise anisolated nucleic acid molecule that encodes the amino acid sequence forthe HCV genotype 1a/1b consensus immunogen of HCV proteins NS3/NS4A or afragment thereof. Some embodiments comprise a recombinant vaccine thatencodes the amino acid sequence for the HCV genotype 1a/1b consensusimmunogen of HCV proteins NS3/NS4A or a fragment thereof. Someembodiments comprise a subunit vaccine that comprises the amino acidsequence for the HCV genotype 1a/1b consensus immunogen of HCV proteinsNS3/NS4A or a fragment thereof. Some embodiments comprise a liveattenuated vaccine and/or a killed vaccine that comprise the amino acidsequence for the HCV genotype 1a/1b consensus immunogen of HCV proteins

Improved vaccines comprise proteins and genetic constructs that encodeproteins with epitopes that make them particularly effective asimmunogens against which anti-HCV immune responses can be induced.Accordingly, vaccines can be provided to induce a therapeutic orprophylactic immune response. In some embodiments, the means to deliverthe immunogen is a DNA vaccine, a recombinant vaccine, a protein subunitvaccine, a composition comprising the immunogen, an attenuated vaccineor a killed vaccine. In some embodiments, the vaccine comprises acombination selected from the groups consisting of one or more DNAvaccines, one or more recombinant vaccines, one or more protein subunitvaccines, one or more compositions comprising the immunogen, one or moreattenuated vaccines and one or more killed vaccines.

According to some embodiments of the invention, a vaccine is deliveredto an individual to modulate the activity of the individual's immunesystem and thereby enhance the immune response. When a nucleic acidmolecules that encodes the protein is taken up by cells of theindividual the nucleotide sequence is expressed in the cells and theprotein are thereby delivered to the individual. Aspects of theinvention provide methods of delivering the coding sequences of theprotein on nucleic acid molecule such as plasmid, as part of recombinantvaccines and as part of attenuated vaccines, as isolated proteins orproteins part of a vector.

According to some aspects of the present invention, compositions andmethods are provided which prophylactically and/or therapeuticallyimmunize an individual

DNA vaccines are described in U.S. Pat. Nos. 5,593,972, 5,739,118,5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055,5,676,594, and the priority applications cited therein, which are eachincorporated herein by reference. In addition to the delivery protocolsdescribed in those applications, alternative methods of delivering DNAare described in U.S. Pat. Nos. 4,945,050 and 5,036.006, which are bothincorporated herein by reference.

The present invention relates to improved attenuated live vaccines,improved killed vaccines and improved vaccines that use recombinantvectors to deliver foreign genes that encode antigens and well assubunit and glycoprotein vaccines. Examples of attenuated live vaccines,those using recombinant vectors to deliver foreign antigens, subunitvaccines and glycoprotein vaccines are described in U.S. Pat. Nos.4,510,245; 4,797,368; 4,722,848; 4,790,987; 4,920,209; 5,017,487;5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424; 5,225,336;5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668; 5,387,744;5,389,368; 5,424,065; 5,451,499; 5,453,364; 5,462,734; 5,470,734;5,474,935; 5,482,713; 5,591,439; 5,643,579; 5,650,309; 5,698,202;5,955,088; 6,034,298; 6,042,836; 6,156,319 and 6,589,529, which are eachincorporated herein by reference.

When taken up by a cell, the genetic construct(s) may remain present inthe cell as a. functioning extrachromosomal molecule and/or integrateinto the cell's chromosomal DNA. DNA may be introduced into cells whereit remains as separate genetic material in the form of a plasmid orplasmids. Alternatively, linear DNA that can integrate into thechromosome may be introduced into the cell. When introducing DNA intothe cell, reagents that promote DNA integration into chromosomes may beadded. DNA sequences that are useful to promote integration may also beincluded in the DNA molecule. Alternatively, RNA may be administered tothe cell. It is also contemplated to provide the genetic construct as alinear minichromosome including a centromere, telomeres and an origin ofreplication. Gene constructs may remain part of the genetic material inattenuated live microorganisms or recombinant microbial vectors whichlive in cells. Gene constructs may be part of genomes of recombinantviral vaccines where the genetic material either integrates into thechromosome of the cell or remains extrachromosomal. Genetic constructsinclude regulatory elements necessary for gene expression of a nucleicacid molecule. The elements include: a promoter, an initiation codon, astop codon, and a polyadenylation signal. In addition, enhancers areoften required for gene expression of the sequence that encodes thetarget protein or the immunomodulating protein. It is necessary thatthese elements be operable linked to the sequence that encodes thedesired proteins and that the regulatory elements are operably in theindividual to whom they are administered.

Initiation codons and stop codon are generally considered to be part ofa nucleotide sequence that encodes the desired protein. However, it isnecessary that these elements are functional in the individual to whomthe gene construct is administered. The initiation and terminationcodons must be in frame with the coding sequence.

Promoters and polyadenylation signals used must be functional within thecells of the individual.

Examples of promoters useful to practice the present invention,especially in the production of a genetic vaccine for humans, includebut are not limited to promoters from Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (MV)such as the BIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV). Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human Actin, human Myosin, human Hemoglobin, humanmuscle creatine and human metalothionein.

Examples of polyadenylation signals useful to practice the presentinvention, especially in the production of a genetic vaccine for humans,include but are not limited to SV40 polyadenylation signals and LTRpolyadenylation signals. In particular, the SV40 polyadenylation signalthat is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to asthe SV40 polyadenylation signal, is used.

In addition to the regulatory elements required for DNA expression,other elements may also be included in the DNA molecule. Such additionalelements include enhancers. The enhancer may be selected from the groupincluding but not limited to: human Actin, human Myosin, humanHemoglobin, human muscle creatine and viral enhancers such as those fromCMV, RSV and EBV.

Genetic constructs can be provided with mammalian origin of replicationin order to maintain the construct extrachromosomally and producemultiple copies of the construct in the cell. Plasmids pVAX1, pCEP4 andpREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr virusorigin of replication and nuclear antigen EBNA-1 coding region whichproduces high copy episomal replication without integration.

In some preferred embodiments related to immunization applications,nucleic acid molecule(s) are delivered which include nucleotidesequences that encode protein of the invention, and, additionally, genesfor proteins which further enhance the immune response against suchtarget proteins. Examples of such genes are those which encode othercytokines and lymphokines such as alpha-interferon, gamma-interferon,platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermalgrowth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18,MHC, CD80, CD86 and IL-15 including IL-15 having the signal sequencedeleted and optionally including the signal peptide from IgE. Othergenes which may be useful include those encoding: MCP-1, MIP-1α, MIP-1p,IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1,MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM., ICAM-1, ICAM-2, ICAM-3,CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L,vascular growth factor, IL-7, nerve growth factor, vascular endothelialgrowth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP,Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, CaspaseICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB,Inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NRB, Bax,TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND,Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F,TAP1, TAP2 and functional fragments thereof.

An additional element may be added which serves as a target for celldestruction if it is desirable to eliminate cells receiving the geneticconstruct for any reason. A herpes thymidine kinase (tk) gene in anexpressible form can be included in the genetic construct. The druggangcyclovir can be administered to the individual and that drug willcause the selective killing of any cell producing tk, thus, providingthe means for the selective destruction of cells with the geneticconstruct.

In order to maximize protein production, regulatory sequences may beselected which are well suited for gene expression in the cells theconstruct is administered into. Moreover, codons may be selected whichare most efficiently transcribed in the cell. One having ordinary skillin the art can produce DNA constructs that are functional in the cells.

In some embodiments, gene constructs may be provided in which the codingsequences for the proteins described herein are linked to IgE signalpeptide. In some embodiments, proteins described herein are linked toIgE signal peptide.

In some embodiments for which protein is used, for example, one havingordinary skill in the art can, using well known techniques, produce andisolate proteins of the invention using well known techniques. In someembodiments for which protein is used, for example, one having ordinaryskill in the art can, using well known techniques, inserts DNA moleculesthat encode a protein of the invention into a commercially availableexpression vector for use in well known expression systems. For example,the commercially available plasmid pSE420 (Invitrogen, San Diego,Calif.) may be used for production of protein in E. coli. Thecommercially available plasmid pYES2 (Invitrogen, San Diego, Calif.)may, for example, be used for production in S. cerevisiae strains ofyeast. The commercially available MAXBAC™ complete baculovirusexpression system (Invitrogen, San Diego, Calif.) may, for example, beused for production in insect cells. The commercially available plasmidpcDNA 1 or pcDNA3 (Invitrogen, San Diego, Calif.) may, for example, beused for production in mammalian cells such as Chinese Hamster Ovarycells. One having ordinary skill in the art can use these commercialexpression vectors and systems or others to produce protein by routinetechniques and readily available starting materials. (See e.g., Sambrooket al., Molecular Cloning a Laboratory Manual, Second Ed. Cold SpringHarbor Press (1989) which is incorporated herein by reference.) Thus,the desired proteins can be prepared in both prokaryotic and eukaryoticsystems, resulting in a spectrum of processed forms of the protein.

One having ordinary skill in the art may use other commerciallyavailable expression vectors and systems or produce vectors using wellknown methods and readily available starting materials. Expressionsystems containing the requisite control sequences, such as promotersand polyadenylation signals, and preferably enhancers are readilyavailable and known in the art for a variety of hosts. See e.g.,Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. ColdSpring Harbor Press (1.989). Genetic constructs include the proteincoding sequence operably linked to a promoter that is functional in thecell line into which the constructs are transfected. Examples ofconstitutive promoters include promoters from cytomegalovirus or SV40.Examples of inducible promoters include mouse mammary leukemia virus ormetallothionein promoters. Those having ordinary skill in the art canreadily produce genetic constructs useful for transfecting with cellswith DNA that encodes protein of the invention from readily availablestarting materials. The expression vector including the DNA that encodesthe protein is used to transform the compatible host which is thencultured and maintained under conditions wherein expression of theforeign DNA takes place.

The protein produced is recovered from the culture, either by lysing thecells or from the culture medium as appropriate and known to those inthe art. One having ordinary skill in the art can, using well knowntechniques, isolate protein that is produced using such expressionsystems. The methods of purifying protein from natural sources usingantibodies which specifically bind to a specific protein as describedabove may be equally applied to purifying protein produced byrecombinant DNA methodology.

In addition to producing proteins by recombinant techniques, automatedpeptide synthesizers may also be employed to produce isolated,essentially pure protein. Such techniques are well known to those havingordinary skill in the art and are useful if derivatives which havesubstitutions not provided for in DNA-encoded protein production.

The nucleic acid molecules may be delivered using any of several wellknown technologies including DNA injection (also referred to as DNAvaccination), recombinant vectors such as recombinant adenovirus,recombinant adenovirus associated virus and recombinant vaccinia.

Routes of administration include, but are not limited to, intramuscular,intranasally, intraperitoneal, intradermal, subcutaneous, intravenous,intraarterially, intraocularly and oral as well as topically,transdermally, by inhalation or suppository or to mucosal tissue such asby lavage to vaginal, rectal, urethral, buccal and sublingual tissue.Preferred routes of administration include intramuscular,intraperitoneal, intradermal and subcutaneous injection. Geneticconstructs may be administered by means including, but not limited to,electroporation methods and devices, traditional syringes, needlelessinjection devices, or “microprojectile bombardment gone guns”.

Examples of electroporation devices and electroporation methodspreferred for facilitating delivery of the DNA vaccines, include thosedescribed in U.S. Pat. No. 7,245,963 by Draghia-Akli, et al., U.S.Patent Pub. 2005/0052630 submitted by Smith, et al., the contents ofwhich are hereby incorporated by reference in their entirety. Alsopreferred, are electroporation devices and electroporation methods forfacilitating delivery of the DNA vaccines provided in co-pending andco-owned U.S. patent application Ser. No. 11/874,072, filed Oct. 17,2007, which claims the benefit under 35 USC 119(e) to U.S. ProvisionalApplication Ser. Nos. 60/852,149, filed Oct. 17, 2006, and 60/978,982,filed Oct. 10, 2007 all of which are hereby incorporated in theirentirety.

The following is an example of an embodiment using electroporationtechnology, and is discussed in more detail in the patent referencesdiscussed above: electroporation devices can be configured to deliver toa desired tissue of a mammal a pulse of energy producing a constantcurrent similar to a preset current input by a user. The electroporationdevice comprises an electroporation component and an electrode assemblyor handle assembly. The electroporation component can include andincorporate one or more of the various elements of the electroporationdevices, including: controller, current waveform generator, impedancetester, waveform logger, input element, status reporting element,communication port, memory component, power source, and power switch.The electroporation component can function as one element of theelectroporation devices, and the other elements are separate elements(or components) in communication with the electroporation component. Insome embodiments, the electroporation component can function as morethan one element of the electroporation devices, which can be incommunication with still other elements of the electroporation devicesseparate from the electroporation component. The use of electroporationtechnology to deliver the improved HCV vaccine is not limited by theelements of the electroporation devices existing as parts of oneelectromechanical or mechanical device, as the elements can function asone device or as separate elements in communication with one another.The electroporation component is capable of delivering the pulse ofenergy that produces the constant current in the desired tissue, andincludes a feedback mechanism. The electrode assembly includes anelectrode array having a plurality of electrodes in a spatialarrangement, wherein the electrode assembly receives the pulse of energyfrom the electroporation component and delivers same to the desiredtissue through the electrodes. At least one of the plurality ofelectrodes is neutral during delivery of the pulse of energy andmeasures impedance in the desired tissue and communicates the impedanceto the electroporation component. The feedback mechanism can receive themeasured impedance and can adjust the pulse of energy delivered by theelectroporation component to maintain the constant current.

In some embodiments, the plurality of electrodes can deliver the pulseof energy in a decentralized pattern. In some embodiments, the pluralityof electrodes can deliver the pulse of energy in the decentralizedpattern through the control of the electrodes under a programmedsequence, and the programmed sequence is input by a user to theelectroporation component. In some embodiments, the programmed sequencecomprises a plurality of pulses delivered in sequence, wherein eachpulse of the plurality of pulses is delivered by at least two activeelectrodes with one neutral electrode that measures impedance, andwherein a subsequent pulse of the plurality of pulses is delivered by adifferent one of at least two active electrodes with one neutralelectrode that measures impedance.

In some embodiments, the feedback mechanism is performed by eitherhardware or software. Preferably, the feedback mechanism is performed byan analog closed-loop circuit. Preferably, this feedback occurs every 50μs, 20 μs, 10 μs or 1 μs, but is preferably a real-time feedback orinstantaneous (i.e., substantially instantaneous as determined byavailable techniques for determining response time). In someembodiments, the neutral electrode measures the impedance in the desiredtissue and communicates the impedance to the feedback mechanism, and thefeedback mechanism responds to the impedance and adjusts the pulse ofenergy to maintain the constant current at a value similar to the presetcurrent. In some embodiments, the feedback mechanism maintains theconstant current continuously and instantaneously during the delivery ofthe pulse of energy.

In some embodiments, the nucleic acid molecule is delivered to the cellsin conjunction with administration of a polynucleotide function enhanceror a genetic vaccine facilitator agent. Polynucleotide functionenhancers are described in U.S. Pat. No. 5,593,972, U.S. Pat. No.5,962,428 and International Application Serial Number PCT/US94/00899filed Jan. 26, 1994, which are each incorporated herein by reference.Genetic vaccine facilitator agents are described in U.S. Pat. No.021,579 filed Apr. 1, 1994, which is incorporated herein by reference.The co-agents that are administered in conjunction with nucleic acidmolecules may be administered as a mixture. With the nucleic acidmolecule or administered separately simultaneously, before or afteradministration of nucleic acid molecules. In addition, other agentswhich may function transfecting agents and/or replicating agents and/orinflammatory agents and which may be co-administered with a GVF includegrowth factors, cytokines and lymphokines such as a-interferon,gamma-interferon, GM-CSF, platelet derived growth factor (PDGF), TNF,epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-10, IL-12 andIL-15 as well as fibroblast growth factor, surface active agents such asimmune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPSanalog including monophosphoryl Lipid A (WL), muramyl peptides, quinoneanalogs and vesicles such as squalene and squalene, and hyaluronic acidmay also be used administered in conjunction with the genetic constructIn some embodiments, an immunomodulating protein may be used as a GVF.In some embodiments, the nucleic acid molecule is provided inassociation with PLG to enhance delivery/uptake.

The pharmaceutical compositions according to the present inventioncomprise about 1 nanogram to about 2000 micrograms of DNA. In somepreferred embodiments, pharmaceutical compositions according to thepresent invention comprise about 5 nanogram to about 1000 micrograms ofDNA. In some preferred embodiments, the pharmaceutical compositionscontain about 10 nanograms to about 800 micrograms of DNA. In somepreferred embodiments, the pharmaceutical compositions contain about 0.1to about 500 micrograms of DNA. In some preferred embodiments, thepharmaceutical compositions contain about 1 to about 350 micrograms ofDNA. In some preferred embodiments, the pharmaceutical compositionscontain about 25 to about 250 micrograms of DNA. In some preferredembodiments, the pharmaceutical compositions contain about 100 to about200 microgram DNA.

The pharmaceutical compositions according to the present invention areformulated according to the mode of administration to be used. In caseswhere pharmaceutical compositions are injectable pharmaceuticalcompositions, they are sterile, pyrogen free and particulate free. Anisotonic formulation is preferably used. Generally, additives forisotonicity can include sodium chloride, dextrose, mannitol, sorbitoland lactose. In some cases, isotonic solutions such as phosphatebuffered saline are preferred. Stabilizers include gelatin and albumin.In some embodiments, a vasoconstriction agent is added to theformulation.

According to some embodiments of the invention, methods of inducingimmune responses are provided. The vaccine may be a protein based, liveattenuated vaccine, a cell vaccine, a recombinant vaccine or a nucleicacid or DNA vaccine. In some embodiments, methods of inducing an immuneresponse in individuals against an immunogen, including methods ofinducing mucosal immune responses, comprise administering to theindividual one or more of CTACK protein, TECK protein, MEC protein andfunctional fragments thereof or expressible coding sequences thereof incombination with an isolated nucleic acid molecule that encodes proteinof the invention and/or a recombinant vaccine that encodes protein ofthe invention and/or a subunit vaccine that protein of the inventionand/or a live attenuated vaccine and/or a killed vaccine. The one ormore of CTACK protein, TECK protein, MEC protein and functionalfragments thereof may be administered prior to, simultaneously with orafter administration of the isolated nucleic acid molecule that encodesan immunogen; and/or recombinant vaccine that encodes an immunogenand/or subunit vaccine that comprises an immunogen and/or liveattenuated vaccine and/or killed vaccine. In some embodiments, anisolated nucleic acid molecule that encodes one or more proteins ofselected from the group consisting of: CTACK, TECK, MEC and functionalfragments thereof is administered to the individual.

The present invention is further illustrated in the following Example.It should be understood that this Example, while indicating embodimentsof the invention, is given by way of illustration only. From the abovediscussion and this Example, one skilled in the art can ascertain theessential characteristics of this invention, and without departing fromthe spirit and scope thereof, can make various changes and modificationsof the invention to adapt it to various usages and conditions. Thus,various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

Each of the U.S. patents, U.S. applications, and references citedthroughout this disclosure are hereby incorporated in their entirety byreference.

Example

Hepatitis C virus (HCV) represents a major health burden with more than170 million individuals currently infected worldwide, equaling roughly3% of the world's population. HCV preferentially infects hepatocytes andis able to persist in up to 70% of infected individuals. It is estimatedthat up to 30% of chronically infected individuals will go on to developprogressive liver disease as a result of HCV infection, making the virusthe leading cause of liver transplantation in the world. Currently thereis no vaccine for HCV. A multi-step approach was used to develop a novelgenotype 1a/1b consensus HCV NS3/NS4A DNA vaccine able to induce strongcellular immunity. This construct is able to induce strong anti-NS3/NS4AT cell responses in C57BL/6 mice, as well as, in Rhesus Macaques.

Materials and Methods

Generation of HCV Genotype 1a/1b Consensus Sequence of NS3/NS4A

The consensus sequence for NS3 was generated from fifteen differentgenotype 1a sequences and twenty-six different genotype 1b sequences.The consensus sequence for NS4A was generated from fifteen differentgenotype 1a sequences and nineteen different genotype 1b sequences. Thesequences were obtained from GenBank, chosen from multiple countries toavoid sampling error and aligned using Clustal X (version 1.8) softwareto generate the final NS3/NS4A consensus sequence.

Additional Modification of the NS3/NS4A Consensus Sequence

The consensus NS3/NS4A sequence was further modified through theaddition of an IgE leader sequence, endoproteolytic cleavage site and aC-terminal HA tag, FIG. 1. GeneOptimizer™ (GENEART, Germany) was used tocodon and RNA optimize the final sequence.

HCV NS3/NS4A DNA Immunogen

The final consensus NS3/NS4A fusion gene (ConNS3/NS4A) was synthesizedand sequence verified by GENEART (Germany). ConNS3/NS4A was digestedwith BamH1 and Not1, and subcloned in to the clinical expression vectorpVAX (Invitrogen) under the control of the CMV promoter. The finalconstruct was named pConNS3/NS4A.

Immunofluorescence

Huh7.0 cells were transiently transfected with pConNS3/NS4A usingLipofectamine™ (Invitrogen) according to the manufacturer's guidelines.After 48 hours of transfection, the cells were permeabilized andexpression of the proteins was determined using a mouse monoclonalantibody to the C-terminal HA tag of the fusion construct (Invitrogen)followed by a TRITC conjugated goat anti-mouse secondary antibody(Invitrogen).

Mouse Studies Immunization/Electroporation

Female 6 to 8 week old C57BL/6 mice were purchased from JacksonLaboratories and were cared for in accordance with the NationalInstitutes of Health and the University of Pennsylvania InstitutionalCare and Use Committee (IACUC) guidelines.

The mice were separated three mice per group and immunized with eitherpConNS3/NS4A or with the empty expression vector pVAX (negativecontrol). Each mouse received three intramuscular injections, two weeksapart. Following each intramuscular injection, a three-electrode arraymade up of 26-gauge stainless steel electrodes was gently inserted intothe muscle and a brief square-wave constant-current EKD wasadministered.

Splenocyte Isolation

The mice were sacrificed one week following the third immunization andthe spleens were pooled according to group. The spleens were crushedusing a Stomacher machine, and the resulting product was put through a40 μM cell strainer to isolate the splenocytes. The cells were treated 5min with ACK lysis buffer (Biosource) to clear the RBCs. Following lysisthe splenocytes were resuspended in RPMI medium supplemented with 10%FBS. The cell number was determined with a hemocytometer.

IFN-Gamma ELISpot

The mouse IFN-gamma ELISpot assays were conducted as previouslydescribed [Yan., J., et al., Enhanced cellular immune responses elicitedby an engineered HIV-1 subtype B consensus-based envelope DNA vaccine.Mol Ther, 2007. 15(2): p. 411-21]. Splenocytes were stimulated with fivedifferent pools of 15mer peptides, overlapping by 8 amino acids andspanning the entire length of the pConNS3/NS4A protein. The peptideswere synthesized by Invitrogen and pooled at a concentration of 2ug/ml/peptide. The splenocytes were plated at a concentration of 200,000cells per well and the average number of spot forming units (SFU) wasadjusted to 1×10⁶ splenocytes for graphing purposes.

Epitope Mapping

The 15mer overlapping peptides were pooled into 21 separate pools, witheach individual peptide represented in two pools of the 21 pools.Splenocytes were then stimulated with each pool in an IFN-gamma ELISpotassay as described above.

Rhesus Macaques Study Study Design and Immunization

A total of five Rhesus Macaques of Indian origin were used andmaintained in accordance with the Guide for the Care and Use ofLaboratory Animals. Plasmids were prepared, HPLC purified (VGXPharmaceuticals, Immune Therapeutics Division, The Woodlands, Tex.) anddiluted in sterile water formulated with 1% (weight/weight)pol-L-glutamate sodium salt (MW=10.5 kDa) (Sigma).

The Macaques were intramuscularly injected followed by electroporationwith the CELLECTRA™ adaptive constant current electroporation device andelectrode arrays. Each monkey was injected with 1 mg of pConNS3/NS4A in0.75 ml volume followed by three pulses of 0.5 Amp constant current,each 1 sec apart and 52 msec in length. Each monkey received twoimmunizations four weeks apart.

Blood Collection and PBMC Isolation

The Macaques were bled once before the first immunization and two weeksfollowing each immunization. The animals were anesthetized with amixture of ketamine (10 mg/kg) and acepromazine (0.1 mg/kg). Blood wascollected in EDTA tubes and PBMCs were isolated using standardFicoll-Hypaque density gradient centrifugation. The isolated PBMCs wereresuspended complete media 1640 with 2 mM/L L-glutamine supplementedwith 10% heat inactivated FBS, 1× anti-biotic/anti-mycotic, and 55 μM/Lβ-mercaptoethanol).

IFN-gamma ELISpot assays were performed as previously described [Boyer,et al., SIV DNA vaccine co-administered with IL-12 expression plasmidenhances CD8 SIV cellular immune responses in cynomolgus macaques. J MedPrimatol, 2005. 34(5-6): p. 262-70] using detection and captureantibodies from MabTech, Sweden.

Results

Construction of a Novel HCV Genotype 1a/1b Consensus Fusion Immunogen ofHCV Structural Proteins NS3/NS4A

Due to the high mutational rate of HCV, designing immunogens withmultiple immune target sites is important not only for protectionagainst various strains of the virus, but for maintaining control inchronically infected individuals by guarding against viral escapemutants. Previous findings report the use of consensus immunogens in thecontext of vaccines may be able to elicit a more broad immune responseas compared to vaccination with the native immunogens alone. Based onthese findings, a construct encoding the NS3/NS4A consensus sequence forHCV genotypes 1a/1b was designed in hopes of increasing the breadth ofthe immune response against the NS3/NS4A proteins. The consensussequence was generated from a total of seventy-five different sequencesobtained from GenBank. Clustal X (version 1.8) software was used tocreate multiple alignments needed to generate a single consensussequence, FIG. 1.

As depicted in FIG. 2, the consensus immunogen was further modified toincrease expression and immunogenicity. NS4A was included within theconstruct due to its reported ability to enhance the stability,expression and immunogenicity of the NS3 protein [Frelin, L., et al.,Low dose and gene gun immunization with a hepatitis C virusnonstructural (NS) 3 DNA-based vaccine containing NS4A inhibitNS3/4A-expressing tumors in vivo. Gene Ther, 2003. 10(8): p. 686-99;Wolk, B., et al., Subcellular localization, stability, andtrans-cleavage competence of the hepatitis C virus NS3-NS4A complexexpressed in tetracycline-regulated cell lines. J Virol, 2000. 74(5): p.2293-304; Tanji, Y., et al., Hepatitis C virus-encoded nonstructuralprotein NS4A has versatile functions in viral protein processing. JViral, 1995. 69(3): p. 1575-81]. An endoproteolytic cleavage site wasintroduced between the two protein sequences to ensure proper folding,as well as, better CTL processing. In addition, to further enhanceexpression of the construct, an IgE leader sequence was fused upstreamof the start codon for the NS3 protein and the entire construct wascodon and RNA optimized for expression in Homo sapiens. The finalsynthetically engineered ConNS3/NS4A gene was 2169 bp in length and wassubcloned into the clinical expression vector pVAX using the BamH1 andNot1 restriction sites.

Expression of pConNS3/NS4A

Expression of pConNS3/NS4A was confirmed through transient transfectionof a Huh7.0 cell line with pConNS3/NS4A, FIG. 3. The translated proteinswere detected with a monoclonal antibody against the C-terminal HA tagof the construct and were visualized using immunofluorescence. As anegative control, cells were also transiently transfected with the emptypVAX vector and stained with a monoclonal anti-HA antibody.

Immunization of C57BL/6 Mice with pConNS3/NS4A Induces Strong CellularNS3- and NS4A-Specific Immune Responses

Following confirmation of the expression of pConNS3/NS4A, mice wereimmunized intramuscularly with the construct, followed byelectroporation, in order to determine whether pConNS3/NS4A could inducecellular immune responses in mice. C57BL/6 received three immunizationsusing four different doses of pConNS3/NS4A. The mice were sacrificed oneweek following the third immunization and cellular immune responses tothe construct were determined using IFN-gamma ELI Spot assays.Splenocytes from vaccinated mice were stimulated with five pools of15mer peptides overlapping by eight amino acids and spanning thesequence of pConNS3/NS4A. As shown in FIG. 4A, pConNS3/NS4A is able toinduce potent cellular immune responses regardless of dose.

Next, using a matrix epitope mapping technique, the dominant epitope ofpConNS3/NS4A was identified in IFN-gamma ELISpot assays. The dominantepitope of pConNS3/NS4A in C57BL/6 mice mapped to peptide 89(LYRLGAVQNEVTLTH—SEQ ID NO:9) located near the C-terminus of the NS3protein, FIG. 4B. This is supported by previous research whichidentified the nine amino acid sequence of GAVQNEVTH—SEQ ID NO:10,contained within peptide 89, as a dominant H-2b CTL epitope. Thisfinding argues for normal and effective processing of our consensusimmunogen.

Immunization of Rhesus Macaques with pConNS3/NS4A Induces StrongCellular NS3- and NS4A-Specific Immune Responses

Although immunogenic in small animal models, DNA vaccines have typicallylost potency when moved into larger animals. However, encouraged by thestrong cellular immune responses elicited by pConNS3/NS4A in mice, wedecided to test the immunogenicity of the construct in a larger animalmodel. Rhesus Macaques were immunized IM/EP with pConNS3/NS4A two times,four weeks apart, FIG. 5A. The animals were bled once before the firstimmunization and two weeks following each immunization. The animals'immune responses to pConNS3/NS4A were determined with IFN-gamma ELISpotassays. FIG. 5B depicts the sum of each monkey's response to all fivepeptide pools, as well as, the average group response for each of thethree time points. Cellular immune responses were not detectablefollowing the prebleed and the first immunization, however, after thesecond immunization the responses increased dramatically to an averageof 555+/−280 SFU/10⁶ PBMCs. Therefore, following only two immunizations,pConNS3/NS4A was able to elicit clear NS3- and NS4-specific cellularimmune responses.

pConNS3/NS4A Elicits a Broad Cellular Immune Response in Rhesus Macaques

Unlike immunization in C57BL/6 mice, where the majority of the immuneresponse was directed at one dominant epitope of pConNS3/NS4A containedwithin a single peptide pool, the majority of immunized monkeys (4 outof 5) were able to elicit strong cellular immune responses to at leasttwo or more peptide pools of pConNS3/NS4A, FIG. 6. Although furtherepitope mapping studies are planned, this suggests that the RhesusMacaques were able to elicit cellular immune responses against multiplesites within the NS3/NS4A proteins. Therefore, the consensus sequence ofpConNS3/NS4A is able to elicit both strong and broad cellular responsesagainst the NS3/NS4A proteins in Rhesus Macaques.

Discussion

It is known that HCV infected individuals who recover from acuteinfection are able to mount early, multi-specific, CD4+ helper and CD8+cytotoxic T-cell responses. It has also been reported that HCV-specificCTL responses are important for control of viral replication inchronically infected individuals [Rehermann, B., et al., Quantitativeanalysis of the peripheral blood cytotoxic T lymphocyte response inpatients with chronic hepatitis C virus infection. J Clin Invest, 1996.98(6): p. 1432-40; Nelson, D. R., et al., The role of hepatitis Cvirus-specific cytotoxic T lymphocytes in chronic hepatitis C. JImmunol, 1997. 158(3): p. 1473-81]. More specifically, a strong T cellresponse against the structural proteins of the virus, in particular theNS3 protein, has been reported to be an important correlate to clearanceof acute infection. Due to the HCV virus's high mutation rate, therelatively conserved structural proteins of the virus, such as NS3, areattractive candidates for T cell based vaccines.

DNA vaccines are well suited for eliciting strong T cell responses.Unlike immunization with recombinant proteins, which tend to produce Th2responses, DNA vaccines are able to induce strong Th1 responses due tothe ability of antigens to be delivered and processed intracellularly.In addition, DNA vaccines theoretically have unlimited boostingcapability and can be readministered as often as desired without fear ofinducing neutralizing antibodies to the plasmid, as is the case withrecombinant viral vectors.

Due to the importance of NS3 specific T cell responses in clearance ofacute infection and control of chronic infection, as well as, the clearadvantages of DNA immunization, a novel HCV DNA vaccine encoding theconsensus sequence of genotype 1a and 1b for the HCV proteins NS3/NS4A(pConNS3/NS4A) was created. Although DNA vaccines are able to elicitstrong cellular immune responses in small animals, the major obstaclefor adoption of DNA vaccine into a clinical setting has been the reducedimmunogenicity of the platform when introduced in larger animal models.Therefore, in order to improve the potency of our construct, amulti-step approach was taken in both its design and administration,including codon and RNA optimization, addition of an IgE leader sequenceand administration of the construct with electroporation; modificationsthat have been shown to increase the expression and immunogenicity ofDNA vaccines.

However, due to the high mutation rate of HCV, a construct able toelicit both strong and broad cellular immune responses targetingmultiple sites within the immunogen may be better able to drive immunityagainst various strains of the virus, as well as, allow for betterpossible control of the virus in infected individuals by guardingagainst viral escape mutants. Previous studies report that incorporationof multiple sequences to create one consensus immunogen is able toproduce broader immune responses. Therefore, as part of our constructdesign we incorporated seventy-five different HCV genotype 1a/1bsequences of the proteins NS3/NS4A in order to create one consensusimmunogen.

The construct, pConNS3/NS4A, is expressed in cell culture, is able toinduce strong NS3- and NS4-specific T cell responses in C57BL/6 micefollowing three immunizations, and is able to elicit both strong andbroad NS3- and NS4A-specific T cell responses in a larger animal model,Rhesus Macaques, following only two immunizations. In fact, one of onlytwo DNA vaccine studies looking at NS3-specific immune responses inducedin Rhesus Macaques. While both studies used similar sequenceoptimization methods, plasmid delivery systems and identical vaccinationschedules, this construct was able to induce much higher NS3 specificimmune responses with fewer immunizations and one-fifth the amount ofDNA as compared to a previous study in which animals received threeimmunizations of 5 mg of DNA [Capone, S., et al., Modulation of theimmune response induced by gene electrotransfer of a hepatitis C virusDNA vaccine in nonhuman primates. J Immunol, 2006. 177(10): p. 7462-71].In addition, pConNS3/NS4A is able to elicit broad responses in RhesusMacaques. Unlike C57BL/6 mice, which responded to one dominant epitopecontained within one peptide pool, the majority of the Rhesus Macaqueswere able to elicit strong cellular immune responses to multiple peptidepools, suggesting that these monkeys are able to mount a response tomultiple epitopes within pConNS3/NS4A.

1. A nucleic acid molecule comprising a nucleotide sequence selectedfrom the group consisting of: SEQ ID NO:1; fragments of SEQ ID NO:1;sequences having at least 90% homology to SEQ ID NO:1; and fragments ofsequences having at least 90% homology to SEQ ID NO:1.
 2. The nucleicacid molecule of claim 1 comprising SEQ ID NO:1.
 3. The nucleic acidmolecule of claim 1 comprising a sequence having at least 95% homologyto SEQ ID NO:1.
 4. The nucleic acid molecule of claim 1 comprising asequence having at least 98% homology to SEQ ID NO:1.
 5. The nucleicacid molecule of claim 1 comprising a sequence having at least 99%homology to a nucleotide sequence selected from the group consisting of:SEQ ID NO:1.
 6. A nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: nucleotide sequences that encodeSEQ ID NO:2; nucleotide sequences that encode an amino acid sequenceshaving at least 90% homology to SEQ ID NO:2; fragments of nucleotidesequences that encode SEQ ID NO:2; and fragments of a nucleotidesequence that encode an amino acid sequence having at least 90% homologyto SEQ ID NO:2.
 7. The nucleic acid molecule of claim 6 comprising anucleotide sequence that encodes SEQ ID NO:2.
 8. The nucleic acidmolecule of claim 1 comprising SEQ ID NO:5.
 9. The nucleic acid moleculeof claim 1 comprising a nucleotide sequence that encodes SEQ ID NO:6.10. The nucleic acid molecule of claim 1 wherein said molecule is aplasmid.
 11. A pharmaceutical composition comprising a nucleic acidmolecule of claim
 10. 12. An injectable pharmaceutical compositioncomprising a nucleic acid molecule of claim
 10. 13. A method of inducingan immune response in an individual against HCV comprising administeringto said individual a composition comprising a nucleic acid molecule ofclaim
 1. 14. The method of claim 13 wherein said nucleic acid moleculeis a DNA molecule.
 15. The method of claim 14 wherein said nucleic acidmolecule is a plasmid.
 16. The method of claim 14 wherein said nucleicacid molecule introduced into the individual by electroporation.
 17. Arecombinant vaccine comprising a nucleic acid molecule of claim
 1. 18.The recombinant vaccine of claim 17 wherein said recombinant vaccine isa recombinant vaccinia vaccine.
 19. A live attenuated vaccine comprisinga nucleic acid molecule of claim
 1. 20. A protein comprising an aminoacid sequence selected from the group consisting of: SEQ ID NO:2,sequences having at least 90% homology to SEQ ID NO:2; fragments of SEQID NO:2; and fragments of sequences having at least 90% homology to SEQID NO:2.
 21. The protein of claim 20 comprising SEQ ID NO:2.
 22. Theprotein of claim 20 comprising a sequences having at least 95% homologyto SEQ ID NO:2.
 23. The protein of claim 20 comprising a sequenceshaving at least 98% homology to SEQ ID NO:2.
 24. The protein of claim 20comprising a sequences having at least 99% homology to SEQ ID NO:2. 25.The protein of claim 20 comprising SEQ ID NO:6.
 26. A pharmaceuticalcomposition comprising a protein of claim
 20. 27. An injectablepharmaceutical composition comprising a protein of claim
 20. 28. Arecombinant vaccine comprising a protein of claim
 20. 29. Therecombinant vaccine of claim 28 wherein said recombinant vaccine is arecombinant vaccinia vaccine.
 30. A live attenuated vaccine comprising aprotein of claim
 20. 31. A method of inducing an immune response in anindividual against HCV comprising administering to said individual acomposition comprising a protein of claim
 20. 32. A method of inducingan immune response in an individual against HCV comprising administeringto said individual a recombinant vaccine of claim
 17. 33. A method ofinducing an immune response in an individual against HCV comprisingadministering to said individual a live attenuated vaccine of claim 19.34. The method of claim 13 wherein the individual has been diagnosed ashaving HCV infection.
 35. A method of inducing an immune response in anindividual against HCV comprising administering to said individual arecombinant vaccine of claim
 28. 36. A method of inducing an immuneresponse in an individual against HCV comprising administering to saidindividual a live attenuated vaccine of claim
 30. 37. The method ofclaim 31 wherein the individual has been diagnosed as having HCVinfection.